Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5′-Monophosphate Decarboxylase

Abstract

The caged complex between orotidine 5′-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5′-monophosphate (FOMP) undergoes decarboxylation ∼300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5′-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5′-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein–phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s–1 for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

Linking coupled motions and entropic effects to the catalytic activity of 2-deoxyribose-5-phosphate aldolase (DERA)

“…hydrogen bonding) interactions that steers substrate selectivity, including stereoselectivity. (2) Extensive experimental work by Richard and co-workers on other enzymes acting on phosphorylated substrates, which include orotidine monophosphate decarboxylase 32 – 34 and triosephosphate isomerase, 35 – 37 shows that remote protein–phosphodianion interactions can, in these cases, be crucial for transition state stabilization, in part by promoting the formation of (and stabilizing of) active high-energy conformations of the protein. (3) In the current case of DERA catalysed aldol cleavage of dR5P, the additional ring opening of the predominantly existing furanose form (99.8% 38 ) of the aldose substrate to the reactive open-chain aldehyde has to be considered (the concentration of the aldehyde in solution is expected to be even lower than 0.2% of the total dR5P, since it will be further equilibrated with the hydrated gem -diol).…”

Role of Loop-Clamping Side Chains in Catalysis by Triosephosphate Isomerase

“…The Y217A mutation results in a large decrease in the rate of loop closure of the phosphodianion, so that this conformational change becomes rate-determining for decarboxylation of OMP. 69 …”

Protein Flexibility and Stiffness Enable Efficient Enzymatic Catalysis

“…When the protein conformational change is uncoupled from the active-site chemistry ( k chem , Scheme 7 ) the protein motions that control the rate constant k c for this conformational change will only limit the value of the kinetic parameter k cat / K m , when k c is rate determining for turnover at low substrate concentrations ( k –c < k chem , Scheme 7 ). 113 , 114 These motions will only limit the value of k cat when they are rate-determining for reactions at saturating [S] ( k ′ –c < k chem ). 113 , 114 The open and closed forms of TIM have been distinguished in solid-state NMR, 41 , 115 , 116 solution NMR, 117 and laser-induced temperature jump fluorescence spectroscopy studies.…”

“… 113 , 114 These motions will only limit the value of k cat when they are rate-determining for reactions at saturating [S] ( k ′ –c < k chem ). 113 , 114 The open and closed forms of TIM have been distinguished in solid-state NMR, 41 , 115 , 116 solution NMR, 117 and laser-induced temperature jump fluorescence spectroscopy studies. 40 The results from studies on the conversion of E O ·S to E C ·S provide evidence that closure of flexible loop 6 over the substrate GAP is partly rate determining for k cat / K m and that opening of this loop to release product DHAP is partly rate determining for k cat for TIM-catalyzed isomerization of GAP ( Figure 1 ).…”